Using primary rat muscle cells, we have been able to specifically block differentiation (fusion) of these myoblasts into muscle fibers. The addition of critical amounts of either ethidium bromide (EtBr) or rifampicin (Rif) blocks differentiation without appreciably changing the cell morphology and is highly reversible upon the removal of the drug. Further, the effect of the drugs is apparently not mediated through mitochondrial protein synthesis, as normal fusion occurs in the presence of chloramphenicol. In this project we propose to: 1) use EtBr and Rif to investigate the early molecular events required for cell fusion and 2) determine the generality of these drugs as inhibitors of differentiation in other systems. The effect of EtBr and Rif on DNA, RNA and protein synthesis in whole cells and in mitochondria will be studied. The relative inhibition of EtBr, Rif, and bromodeoxyuridine (BrdU) will also be compared; BrdU is the only other known specific inhibitor of differentiation. The molecular mode of action of EtBr and Rif will be examined, by searching for RNA of mitochondrial origin appearing outside the mitochondria. Our preliminary work indicates this as a possible mechanism controlling differentiation. Also, the existence of genes involved in differentiation as episomes and/or the role of a procaryotic-like RNA-polymerase outside the mitochondria will be investigated. The generality of EtBr and Rif as specific inhibitors of differentiation will be investigated in other systems sensitive to BrdU. Also, systems such as sea urchin development which are very general and have been well studied, will be tested.